Bacillus subtilis produces a wide array of extracellular metabolites that can inhibit the growth of bacteria and fungi or modify their behavior to attenuate the production of antibacterial products by potentially dangerous neighbors. We here propose to use the new technique of imaging mass spectrometry and classical analytical chemistry to systematically identify the extracellular metabolome of B. subtilis, with a focus on characterizing the interactive metabolome that is induced by other bacterial species. We will investigate the role these compounds play in two distinct outcomes of the interaction of B. subtilis with other species. The first is an impasse, in which B. subtilis forms closely abutting colonies with other species that produce a variety of antibacterial compounds (such as P. aeruginosa). The second, more frequent behavior is contact-dependent predation, in which B. subtilis moves towards, invades and destroys neighboring colonies, leading to death of the prey species and expanding the territory of the B. subtilis colony. These reproducible behaviors are conserved in different undomesticated B. subtilis strains. We will determine if these behaviors depend on the interactive metabolome and investigate the effects individual compounds have on target cell viability and behavior. We will further investigate the genetic requirements for interspecies interactions to identify stress responses, developmental and biosynthetic pathways that contribute to these distinct outcomes and we will use fluorescence microscopy to visualize the cellular consequences of interspecies interactions. These studies will illuminate the mechanistic basis for interspecies interactions and identify secondary metabolites that affect viability or behavior of other species that represent potential new antibacterial drugs. PUBLIC HEALTH RELEVANCE: Bacteria produce many extracellular metabolites that mediate their interaction with other species, many of which have antibacterial and antifungal activities. We will here elucidate the chemical, genetic and cellular mechanisms by which these molecules allow Bacillus subtilis to interact with other bacterial species, producing outcomes ranging from coexistence to the invasion and destruction of neighboring colonies. Interspecies interactions are critical in medicine and the metabolites that facilitate destruction of other species represent promising new pharmaceutical leads.